Insulin-like growth factor-1 (IGF-1) is a polypeptide of 70 amino acids with a molecular weight of 7648 daltons. This single chain protein has three intrachain disulfide bridges. These disulfide bonds, along with numerous hydrogen bonds and hydrophilic interactions, maintain the compact tertiary structure of this molecule. However, it has been shown that, upon reduction and reoxidation, IGF-1 can refold in a variety of ways, forming as many as 15 monomeric configurations [see Meng, et al., J. Chrom. 433:183 (1988)]. Consequently, attempts to produce large quantities of this peptide can lead to the formation of a complicated mixture of product forms which must be purified for further use.
Insulin-like growth factor-1 belongs to a heterogeneous family of peptides which share some of the biological and chemical properties of insulin, but which are antigenically distinct from insulin. Currently available experimental evidence suggests that IGF-1 promotes growth by mediating the effects of growth hormone. Thus, such processes as skeletal growth, cell replication and other growth related processes are affected by IGF-1 levels. Physiological concentrations of IGF-1 have been shown to be influenced by such conditions as thyroid disease, diabetes and malnutrition [see Preece, in Horm. Blood, 4: 108 (1983)].
IGF-1 has also been shown to act synergistically with other growth factors, for example, in accelerating the healing of soft and mesenchymal tissue wounds [see Lynch et al., in J. Clin. Periodontol., 16: 545 (1989) and Lynch et al., in Proc. Natl. Acad. Sci. USA, 84: 7696 (1987)], and in enhancing the growth of mammalian cells in serum-free tissue culture medium [see Burleigh and Meng, in American Biotech. Lab., 4: 48 (1986)].
Considering the many clinical and research applications of IGF-1, a ready supply of IGF-1 will be of great value to the medical and biotechnology fields. Since isolation from natural sources is technically difficult, expensive, and time consuming, recent efforts have centered on the development of efficient recombinant methods for the production of IGF-1.
The methylotrophic yeast Pichia pastoris has recently been developed as an improved host for the production of recombinant products. Recombinant Pichia pastoris strains have been shown to be capable of secreting certain recombinant proteins in the gram per liter range. In addition, such strains have been shown to be capable of adapting to fed batch or continuous cultivation fermentation conditions. Moreover, such strains have an extremely stable recombinant phenotype and are capable of maintaining high yields of the desired recombinant expression product over several orders of fermentation scale. Indeed, Brierley, et al., in copending application U.S. Ser. No. 578,728, filed Sep. 4, 1990, have recently shown that P. pastoris is an excellent host for the recombinant production of IGF-1. The disclosure of this copending application is hereby incorporated by reference in its entirety. In view of the availability of medium containing high levels of recombinantly produced IGF-1, there is needed an efficient means for the recovery and purification of IGF-1 from such medium.
Recombinantly produced IGF-1 frequently consists of a mixture of several different forms of IGF-1, i.e., intact, monomeric, correctly-folded material (also referred to herein as authentic IGF-1), as well as various aberrant forms, such as, misfolded material (i.e., having improperly formed disulfide bonds), nicked material (i.e., wherein one or more of the peptide bonds of the amino acid backbone have been broken, but the molecular weight of the resulting species is substantially the same as that of intact material, since the nicked material has the same number of amino acid residues as intact material, and the fragments of nicked material are held together by disulfide bonds), cleaved material (e.g., wherein one or more peptide bonds are broken so that two fragments of lower molecular weight, relative to intact material, are produced; or peptide lacking one or more amino acid residues relative to intact material), multimeric forms (i.e., dimers, trimers, etc., wherein disulfide bonds are formed between two or more different IGF-1 monomer chains), and so on. Due to the substantial similarity of the various forms of IGF-1, the purification of recombinantly produced material presents a difficult technical challenge. Not only does such purification require the separation of IGF-1 peptides from the other peptides produced during fermentation, in addition, a separation is required which is selective enough to distinguish between the various forms of IGF-1 which may be present.
Brierley et al. also describe in co-owned international application No. PCT/US91/06452, which is a continuation-in-part of U.S. Ser. No. 578,728 filed with the Patent Cooperation Treaty on Sep. 4, 1991, production of IGF-1 in P. pastoris strains deficient in proteolytic activities that can degrade the recombinant product to yield aberrant forms such as nicked IGF-1. The use of protease-deficient strains of P. pastoris as hosts for recombinant expression of heterologous proteins susceptible to degradation by P. pastoris proteases is described in U.S. Ser. No. 07/678,916 filed Apr. 1, 1991. The disclosure of these co-pending applications is hereby incorporated by reference in their entirety. Fermentations of IGF-1-producing P. pastoris strains deficient in proteolytic activity yielded 50-100% more authentic IGF-1 and 30% less nicked IGF-1 than similar fermentations of IGF-1-producing P. pastoris strains that were not deficient in proteolytic activity. Although purification of authentic IGF-1 from the broth of a P. pastoris strain deficient in proteolytic activity might be facilitated by the lower amounts of nicked IGF-1 in the broth, a separation process capable of distinguishing between the various forms of IGF-1 is still required for such purification